Method: 120 male Sprague Dawley (SD) rats were randomly divided into four groups: group A, group N, group B and group C. Group A, group B and group N underwent collagenase-induced Intracerebral Haemorrhage (ICH) surgery procedure as animal model groups. Group C received a 10 μl saline injection with the same surgical procedure as the control group. US-guided transcranial drilling and catheter drainage were performed on group A. CT-controlled transcranial drilling and catheter drainage were performed on group B. Then group A, group B and group N were evaluated by ultrasound and CT. All groups were observed and assessed the behavior tests and mortality for 4 weeks. The remains of all groups were put to death after assessment. Apoptosis of neuron cells were identified by hematoxylin and eosin stain and immunohistochemistry.

Results: The mortality of each group showed no significant difference (p>0.05). Behavioral testing showed that there was no significant difference between group A and B (p>0.05). The expression of NFkB and caspase-3 revealed that there was a few apoptosis of neuron cells both in group A and B.

Conclusions: US-guided transcranial drilling and catheter drainage are as accurate as CT-controlled transcranial drilling and catheter drainage. It could result in a significant reduction of procedure expenditure under the avoidance of radiation and show the same therapeutic effect.

Keywords

Introduction

Intracerebral hemorrhage (ICH) is a common and potentially
disabling event worldwide. There is an approximate 50%
mortality rate during the month following ICH [1]. High
morbidity after ICH would impact the patients’ quality life for
a long time. The understanding of cerebrovascular events
during a brain hemorrhage is particularly important for the
effective medications and treatments [2]. Computed
tomography (CT) and magnetic resonance imaging (MRI) are
well-established techniques that are used in clinical imaging
investigations of hematoma area and development [3-5].
However, the techniques are too large to carry. Considering
ultrasound is the most widely used technique for detecting
hematoma in the early stage [6], our current study aimed to use
ultrasound technique for the investigation of cerebrovascular
events during a brain hemorrhage in a collagenase-induced
ICH in rats. We want to find out one quicker and accurate
treatment for early ICH, and simpler and more feasible
examination for delay ICH.

Materials and Methods

Animal model

Animals were housed and treated in accordance with Chinese
national guidelines for the care of laboratory animals and was
approved by the Animal Care and Use Committee of the
Chinese Academy of Military Medical Science.

A total of 120 young male Sprague Dawley (SD) rats
(Laboratory Animal Center, Academy of Military Medical
Sciences, Beijing, China) were used in this study. Animals
weighing 250-280 g, aged 4-7 months, were housed individually with a 12 h light/dark cycle and given access to
food and water ad libitum in a temperature-controlled
environment (20-22°C), randomly divided into four groups:
group A, group N, group B and group C. The performance of
all groups was listed as follows.

Collagenase-induced ICH surgery procedure

Group A, group B and group N underwent collagenase-induced
ICH surgery procedure as animal model groups. The protocol
of collagenase-induced ICH was modified based on an
established method [7]. Briefly, animals were anesthetized with
10% chloral hydrate (0.4 mg/kg for induction and 0.02 mg/kg
for maintenance) and placed into a head-holder. Maintained the
body temperature at 37°C by a heating lamp to prevent
hypothermia during the surgery. Blood pressure,
electrocardiography (ECG), blood pH PCO2 and PO2 were
monitored and maintained within normal physiological limits.
Group A, group B and group N were infiltrated with 1%
lidocaine at the scalp area, explored a midline scalp incision. A
burr hole was drilled through the skull. The injection sites were
3 mm right to midline, 1 mm posterior to the anterior
fontanelle, and 5 mm below the surface of the skull.
Collagenase (IV; Sigma, America) dissolved in saline was
infused over 2 min. After infusion, the hole was sealed with
bone wax. The wound was sutured, and the animal was then
transferred to machine lab for the first scanning after surgery.
After evaluated of bleeding site and severity, group A
underwent US-guided transcranial drilling and catheter
drainage therapy. Group B underwent CT-controlled
transcranial drilling and catheter drainage. Group A, group B
and group N were evaluated the bleeding part and severity by
ultrasound and CT. Group C received a 10 μl saline injection
with the same surgical procedure as the control group.

The Bederson score was calculated as follows: Rat tail was
lightly grasped 10 cm above the desktop, and the score was
graded as the following: 0, no neurological deficit; 1, flexion of
the contralateral wrist and elbow and flexion adduction of the
contralateral shoulder; 2, signs in score 1 plus reduced
resistances when pushing toward the paralyzed side; 3, circling
toward the paralyzed side when acting (tail-chasing like) [8].

The beam-walking score was graded as the following: rats
were placed 10 cm above the ground on an 80 × 2.5 × 2.5 cm
stick to be allowed to walk, and the scores were graded as
follows: 0, jump onto the balance beam and walk without
falling; 1, can jump onto the balance beam and walk with less
than 50% chances of falling; 2, can jump onto the balance
beam and walk with more than 50% chance of falling; 3, can
jump onto the balance beam with help from the ipsilateral hind
limbs but cannot move forward due to the paralyzed
contralateral hind limbs; 4, cannot walk on the balance beam
but can sit on it; and 5, fall after being placed on the balance
beam [10].

All groups were observed the mortality for 4 weeks. The
remains of all groups were put to death at the 4 weeks after
ICU.

Hematoxylin and eosin staining (HE staining)

At 4 weeks after ICU, after deeply anesthetized and perfused
with 4% paraformaldehyde, the remains were placed in supine
position. Blood and cerebrospinal fluid samples were draw and
stored in a cryostat at -80°C for the later surgery. The skull was
opened, and the whole brain was dissected. After removing the
cerebellum, the brain sample was placed into the same fixative
solution and fixed for another 24 h, embedded in paraffin, and
finally cut into 4 μm thickness serial sections. Hematoxylin
and eosin stain in accordance with the general steps to
complete.

The amplification process of NF-kB: predenaturation 5 min
at 94°C, then 30 cycles of denaturation 30 s at 94°C annealing
30 s at 60°C, and elongation 40 s at 72°C, lastly with
elongation 10 min at 72°C. The amplification process of bactin:
predenaturation 5 min at 94°C, then 30°C cycles of
denaturation 30 s at 94°C, annealing 30 s at 55°C, and
elongation 40 s at 72°C, lastly with elongation 10 min at 72°C.
The results were referred to β-actin, electrophoresed in 2%
agarose-gel, analyzed by gel image machine, then quantified
and saved by photograph. The index number of mRNA
(RI)=the mRNA scanning value/β-actin mRNA scanning
value.

Statistical analysis

Data were expressed as mean ± standard deviation (Mean ±
SD). All data analysis was performed by SPSS 20.0. ANOVA
was used for multiple-group comparison with post hoc Tukey
test. P<0.05 was considered statistically significant.

Image analysis

Under the microscope (10 × 40), NF-kB +/caspase-3+ nuclei
close to the hematoma were captured in 5 randomly selected
sections from each animal by Olympus Microscope IX73, and
analyzed by Image Analysis Software (Image J 1.29X).

Results

There are 120 rats before the study and 8 were dead after the
ICH procedure (2 in group A at 1 day after surgery, 2 in group
B at 1 day after surgery, 4 in group N within 3 days after ICH
procedure). The survival rate of group A was 93.33%, that of
group B was 93.33%, that of group N was 90%, that of group
C was 100%. The behavioral tests of all groups were listed in Table 1 and Figure 1. Group A, B and N as collagenaseinduced
ICH group exhibited a significant higher scores in the
Bederson test, the Longa test and the beam-walking test
compared to group C as control group (p<0.05). The
behavioral scores of group A exhibited no significant
difference in the Bederson test, the Longa test and the beamwalking
test compared to group B (p>0.05). The behavioral
scores of group A and group B exhibited significant lower
scores in the Bederson test, the Longa test and the beamwalking
test compared to group N (p<0.05).

HE staining revealed that neuron cells near the needle track in
group N were replaced by several sizes of cavities, glial cells
and collagen fibers around the cavities formed glial scar
(Figure 2A). HE staining revealed that a few neuron cells near
the needle track in group A and B were hypertrophy and the
branch were reduced (Figure 2B and Figure 2C). While in
group C the neuron cells arranged in order and norm morph
(Figure 2D). Immunohistochemistry revealed that NF-kB and
Caspase-3 showed the most positive expression in group N,
while that showed the numbers were reduced in group A and
B. There was no NF-kB or Caspase-3 expressed in group C.

Figure 2: HE staining of the brain sample in all groups.

The mRNA expression levels of NF-kB and Caspase-3 were
similar with the result of immunohistochemistry. The mRNA
expression levels of NF-kB and Caspase-3 in group A were
similar with that in group B, while were significantly higher
than that in group C, lower than that in group N (Table 2).

Group A

Group B

Group N

Group C

NF-kB

0.45 ± 0.23

0.43 ± 0.31

1.07 ± 0.17

0.02 ± 0.01

Caspase-3

0.46 ± 0.18

0.47 ± 0.21

1.04 ± 0.08

0.01 ± 0.01

Table 2: The association between NF-kB and Caspase-3 of all groups.

Discussion

Our study consisted on histological characterization and
behavioral testing of our ICH model associated with the
therapy of ICH up to 2 months. The results extended our
knowledge of neuron cells changes during haemorrhage and
pioneered a new technique in collagenase-induced ICH animal
model [11,12]. ICH is the most commonly produced in rat by
either infusing autologous blood or collagenase [13]. In this
study, we chose collagenase IV to induce ICH for the
obviously neurological deficit and a high persistence [14]. In
our study, neurological deficit caused by hematoma were
observed in group A, B and N, especially in group N. It could
see that the behaviour tests of group N had higher scores and
gentler downward trend compared to group A and B.
Combined with results of HE staining and
immunohistochemistry, it showed that ICH would cause the
irreversible damage of brain tissue and neuronal apoptosis. The
faster appropriate therapy is given, the better recover we get
[15]. The mortality of group N was the highest in the animal
model groups, as there was no any therapy for group N after
ICH procedure. We compared the effect of US-guided and CTcontrolled
transcranial drilling and catheter drainage on
collagenase-induced ICH in rats, the mortality showed that
there was no significant difference between group A and B.
Ultrasound could be the new diagnostic and therapeutic
technique and extend in the clinical application for ICH [16].

For further studying the brain damage of each group, we
analysed the apoptosis of neuronal cells in each group. Many
reports have proved that haemorrhage would induce delayed
brain injury even when the haemorrhage was removed [17].
The apoptosis of neuronal cells was associated with the
bleeding and persistence time of haemorrhage [18]. Previous
reports suggested that the expression of NF-kB and caspase-3
were associated with the injury of granule cell [19,20]. NF-kB
exists in cerebrovascular endothelial cells, neuronal cells and
glial cells and would be activated by ischemia of the central
nervous system [21]. It hypothesized that NF-kB involved in
regulating gene transcription function, mediated Fas associated
death domain protein (FADD), activated Caspase-3 and finally
induced apoptosis [22,23]. Caspase-3 (named cysteine protease
32) which is one of the most important caspase, also belongs to
interleukin-1-β convertase (ICE) family [24], is relevant to
apoptosis of neuronal cells after intracerebral haemorrhage
[25,26]. Caspase-3 plays an important role in protein
hydrolysis as downstream effect enzyme and induces
functional proteins cracking and apoptosis [27,28]. In this
study we found that Caspase-3 and NF-kB had high expression
near the hemorrhage even it had been removed for weeks. It
implied that the injury would sustain after the therapy. While
As the mechanism of delay injury is complicated, we would
continue to study the mechanism and find a better way to
reduce the delay injury, for improving the quality life of
patients.